Transforming growth factor-alpha (TGF-alpha ) and epidermal growth factor receptor (EGFR) immunoreactivities were examined in the cerebral cortex of the rat during postnatal development. TGF-alpha -immunoreactive cells were found at birth in all cortical layers except the molecular layer. TGF-alpha -immunoreactive cells were most abundant in the parietal cortex at P20. The intensity and number of the TGF-alpha -immunoreactive cells increased at postnatal days 15 or 20 (P15 - P20). Mature patterns of TGF-alpha -immunoreactive cells were achieved at P20. EGFR -immunoreactive cells appeared only in dorsal endopiriform cortex at P0. The first EGFR -immunoreactive cells were observed in the neocortex at P3. These cells were most abundant in the parietal cortex at P90. In adult, the most prominent EGFR immunoreactivity occured in layer IV, V and VI. These cells were numerous in the frontal and parietal cortex, diminishing laterally towards the insular cortex. Adult patterns were reached on and after P10. The time of appearance and localization of EGFR immunoreactivity correlated with functional activity in the different cortical areas. No clear labelling of glial cells with TGF-alpha and EGFR antibodies was found. TGF -alpha and EGFR immunoreactivity was observed in the majority of neurons in the postnatal developing and adult cerebral cortex of the rat. Also double -immunohistochemistry with antibodies to TGF-alpha and EGFR showed co-localization of TGF -alpha and EGFR in neurons of the cerebral cortex. Co-localization of TGF-alpha and EGFR was first detectable in most cortices at P3. By P10, these neurons showed immature neuronal features. The present results showing TGF -alpha and EGFR immunoreactivity is widely distributed in the postnatal developing (except P0) and adult cerebral cortex, mainly localized in neurons. And TGF-alpha and EGFR co-localize in most neurons, thus indicating that most EGFR -containing cells are TGF-alpha -synthesizing cells. In addition to difference of time of appearance and mature neuronal pattern suggest that TGF-alpha has the capacity of activating the EGFR in the normal postnatal developing cerebral cortex, therefore, TGF-alpha and EGFR may interact within cortical neurons through many different mechanism according to postnatal age.
Adult ; Animals ; Antibodies ; Cerebral Cortex* ; Epidermal Growth Factor* ; Humans ; Immunohistochemistry ; Neocortex ; Neuroglia ; Neurons* ; Parturition ; Rabeprazole ; Rats* ; Receptor, Epidermal Growth Factor ; Transforming Growth Factor alpha

Transforming growth Factor-alpha(TGF-alpha) and epidermal growth factor (EGF) are polypeptides which interact with the epidermal growth factor receptor (EGFR) to produce its biological effects. In normal human tissues, its immunocytochemical presence and biological effects have been demonstrated. The aim of the present investigation was to elucidate the immunolocalization of TGF-alpha and EGF in melanocytic nevi. The data presented in this paper focus attention on comparison of TGF-alpha and EGF in intradermal nevus. The expression of TGF-alpha was stronger than EGF in epidermis of melanocytic nevi. In intradermal nevus, TGF-alpha immunoreactivities were present in most of layers of epidermis and a group of nevus cells in dermis were uniformly immunoreactive with minimal cytoplasm. The expression of EGF was found in part of cytoplasm of keratinocytes in the stratum spinosum and stratum granulosum of epidermis. However, keratinocytes in the stratum basale and nevus cells did not demonstrate immunoreactivity for EGF. In epidermal appendages, the staining intensity of EGF was generally weaker than for TGF-alpha except cells in the external root sheath of hair follicle. In conclusion, some regional variations in the intensity of the immunostaining between TGF-alpha and EGF were present in melanocytic nevi. Our results indicate that TGF-alpha may play a role in growth and proliferation of nevus cells.
Cytoplasm ; Dermis ; Epidermal Growth Factor* ; Epidermis ; Hair Follicle ; Humans ; Immunohistochemistry ; Keratinocytes ; Nevus ; Nevus, Intradermal ; Nevus, Pigmented* ; Peptides ; Receptor, Epidermal Growth Factor ; Transforming Growth Factor alpha

Degenerate oligonucleotide primed PCR is an useful technique to amplify whole genome and its the applications for fluorescent in situ hybridization and comparative genomic hybridization (CGH) were reported. For the CGH, topoisomerase and sequenase were recommended to use for the better hybridization. But adding the enzymes to PCR reaction per every cycle is labor-intensive and can easily contaminate PCR reaction. This study was carried out to prove the possibility of application of DOP-PCR to CGH without use of sequenase. Several combinations of CGH e.g., DOP-PCR amplified normal DNA vs. DOP-PCR amplified normal DNA, DOP-PCR amplified normal DNA vs. non-DOP normal DNA, DOP-PCR amplified normal DNA vs. DOP-PCR amplified MCF-600 cell line DNA, and non-DOP normal DNA vs. non-DOP MCF-600 DNA were performed. In addition, randomly selected microsatellite loci were tested to know whether DOP-PCR covers whole genome amplification. Apparently the DOP-PCR provides enough amount and size of DNA for CGH application and covers whole genome amplification. These results suggest that DOP-PCR can be used for CGH and genotyping.
Cell Line ; Comparative Genomic Hybridization* ; DNA ; Genome ; In Situ Hybridization, Fluorescence ; Microsatellite Repeats ; Polymerase Chain Reaction*

Normal angiogenesis is very important in the embryonic period and even in the postnatal period. If the vascularization is inadequate or excessive, several pathologic conditions may develop. Angiogenesis is well controlled and there are several angiogenic factors. Vascular endothelial growth factor (VEGF) has been identiified as an endothelial cell specific mitogen with potent angiogenic properties. Recently, a matricellular protein known as SPARC (secreted protein, acidic and rich in cysteine) is also proposed as one of new angiogenic factors. So, I planned to investigate the expression pattern of these two factors in the rat retina and to correlate their expression pattern with intraretinal vascularization. I used Sprague-Dawley rat as the experimental animals and divided them into 7 groups according to their postnatal age: 0-day, 3-day, 7-day, 14-day, 21-day, 28-day and adult. Immunohistochemistry was done. The results were as follows. 1. No SPARC immunoreactivity is observed in neural retina until postnatal day 7. At that time, weak immunore-activity is noted and it is gradually increased. The most intense immunoreactivity is noted at postnatal day 21 and 28. SPARC immunoreactivity is restricted in the nerve fiber layer and especially prominent around the blood vessels. Although SPARC immunoreactivity is much weaker than that of GFAP, the expression patterns of both factors are similar to each other. But, no SPARC immunoreactivity is observed in the adult retina. 2. Weak VEGF immunoreactivity is observed in the nerve fiber layer, ganglion cell layer and inner portion of inner nuclear layer even at postnatal day 0. After postnatal day 14, VEGF immunoreactivity is dramatically decreased in the nerve fiber layer, but it still remained in the ganglion cell layer and inner portion of inner nuclear layer. Although immunoreactivity is most intense at postnatal day 14 and 21, VEGF immunoreactivity is still observed in the ganglion cells at postnatal day 28 and adult. The spatial and temporal patterns of VEGF expression suggest that VEGF may function as a direct angiogenic factor in the retinal angiogenesis of rat. In the contrary, SPARC immunoreactivity is observed transiently in some period. So, SPARC is seemed to be a regulator that is involved only in limited steps of the retinal angiogenesis.
Adult ; Angiogenesis Inducing Agents ; Animals ; Blood Vessels ; Endothelial Cells ; Ganglion Cysts ; Humans ; Immunohistochemistry ; Nerve Fibers ; Rats* ; Rats, Sprague-Dawley ; Retina* ; Retinaldehyde ; Vascular Endothelial Growth Factor A*

The tottering (tg/tg) is neurologic mutant mouse exhibiting three neurological disorders: ataxia, petit mal-like absence seizures and myoclonic intermittent movement disorder. The tottering mouse carries an autosomal recessive single gene mutation on chromosome 8. The leaner (tgla) and Nagoya rolling (tgrol) are another two alleles of the tottering (tg). The combination of two mutant (tottering and leaner) produces compound heterozygous, tottering/leaner (tg/tgla) mouse. The genetic etilogy of the tottering and leaner was identified to be a mutation in voltage-dependent calcium channel a1A subunit. It made us link these animal model to human neurologic disease such as autosomal dominant cerebellar ataxia (SCA6), familial hemiplegic migraine and episodic ataxia type-2. The different onset and severity of neurological symptom of these three mutants (tg/tg, tg/tgla, tgla/tgla) offer good scale to analysis of pathophysiolgy of the neurologic disorder. Altered synapase between parallel fiber varicosity and dendritic spines of Purkinje cell was observed in adult tottering and leaner mice. Through the electron microscopic observation and anticalbindin-28 kd immunohistochemistry, we anaylzed not only the relationship between neurologic symptoms and synaptic plasticity around the ataxic onset of tottering, leaner and tottering leaner double mutation but also Purkinje cell morphology affected by voltage-sensitive calcium channel a1A subunit mutation in totterring mouse. Purkinje cell dendritic spines from proximal dendrites and axonal swellings of Purkine cell were observed frequently in wild type mice. The first apperance point of altered synapse based on semi-quantitative analysis was postnatal 15 days in leaner, postnatal 18 days in totering/leaner double mutation, and 30 days in tottering. These data suggest that altered synapse is associated with ataxia in tottering and leaner mice. Further study is needed to determine whether altered synapse is primary cause of ataxia.
Adult ; Alleles ; Animals ; Ataxia ; Axons ; Calcium Channels ; Cerebellar Ataxia ; Cerebellum* ; Chromosomes, Human, Pair 8 ; Dendrites ; Dendritic Spines* ; Epilepsy, Absence ; Humans ; Immunohistochemistry ; Mice* ; Mice, Neurologic Mutants ; Migraine with Aura ; Models, Animal ; Movement Disorders ; Nervous System Diseases ; Neurologic Manifestations ; Plastics* ; Synapses*

Nitric oxide (NO), a free radical that has been postulated to act as a neurotransmitter, neuromodulator, or second messenger molecule in nervous system, is synthesized from L-arginine by nitric oxide synthase (NOS). The NADPH-diaphorase (NADPH-d) histochemical techenique provides a simple and robust method to stain the selected populations of NOS neurons in the brain. This study was aimed to clarify the distribution of NOS neurons in the brain stem of rats. To verify the distribution of NOS neurons in the brain stem, the neurons were stained by the NOS immunohis-tochemistry and NADPH-d histochemistry. Image analyzer-assisted densitometry and cell counting method have been used to quantitatively characterize groups of neuronal cells. Double labeling of NOS immunohistochemistry and NADPH-d histochemistry showed the coexistence of NOS and NADPH-d in same neurons. Neuronal cell bodies exhibiting NOS/NADPH-d staining were found in particular nuclei throughout the brain stem. The number of NOS/NADPH-d neurons were variable in brain stem nuclei. The NADPH-d neurons exhibited different intensities of reaction product. Some groups, including paradorsal raphe nucleus, laterodorsal tegmental nucleus and pedunculopontine tegmental nucleus were extremely heavily stained. Other neurons such as those in the interpeduncular nucleus, central gray, substantia nigra lateralis, nucleus solitarius and raphe obscurus nucleus were moderately stained, while other neurons such supragenual nucleus, lateral paragigantocellular nucleus and prepositus hypoglossal nucleus were weakly stained. The present study describes the many locations within the brain stem in which NADPH-d occurs. Since NADPH-d activity colocalizes with NOS, the results indicate the likely involvement of nitric oxide in the neuronal functions of many brain stem nuclei of rats.
Animals ; Arginine ; Brain Stem* ; Brain* ; Cell Count ; Densitometry ; Immunohistochemistry ; Nervous System ; Neurons ; Neurotransmitter Agents ; Nitric Oxide Synthase* ; Nitric Oxide* ; Pedunculopontine Tegmental Nucleus ; Raphe Nuclei ; Rats* ; Second Messenger Systems ; Solitary Nucleus ; Substantia Nigra

The cholinergic neurons in the striatal complex are the major interneurons that integrate the informations incoming to and outflowing from the striatum. The shape of synapses may change even after birth and the synaptic morphology reflects the functional state of synapse. However, it is not well known about the synaptic morphology of the mouse striatal cholinergic neurons in their early postnatal life. Thus, we investigated the synaptic morphology of the mouse striatal cholinergic neurons in their early postnatal life by the electron microscopy combined with immunohis-tochemistry. In addition, we investigated the trends of change in synaptic morphology and whether the difference between two compartments exists or not. Experimental animals which are ICR mice, were divided into 5 groups according to their postnatal age: 3-day, 1-week, 2-week, 4-week, and 6-week. Pre-embedding immunohisto-chemistry was done with anti-choline acetyl transferase antibody. The results were as follows. 1. In synapses that immunoreactive terminals constitute the presynaptic components, most of synapses are symmetric type in all age groups (p<0.05). Most of synapses in the dorsal striatum are symmetric form from 1-week of postnatal age, but it is not prominent in the ventral striatum until 2-week of postnatal age. 2. In synapses that immunoreactive terminals constitute the postsynaptic components, both symmetric and asymmetric synapses are noted in similar proportions (p<0.05). There are no difference in the synaptic morphology between dorsal and ventral striatum. 3. No specific findings are observed in synaptic curve according to the postnatal age or compartment. In conclusion, the synaptic morphology of mouse striatal cholinergic neurons is similar to mature pattern from 2-week of postnatal age. And it is thought that period between birth and 2-week of postnatal age is the critical period for synaptogenesis. The synaptic curve does not reflect the degree of synaptic maturity. Further investigations will be required to generalize the synaptic curve as a marker for synaptic maturity.
Animals ; Basal Ganglia ; Cholinergic Neurons* ; Critical Period (Psychology) ; Humans ; Immunohistochemistry ; Interneurons ; Mice* ; Mice, Inbred ICR ; Microscopy, Electron ; Parturition ; Synapses ; Transferases

To investigate the distribution, ultrastructure and synapsis of serotoninergic cells and CGRP nerve fibers in mammalian taste buds, immunohistochemistry and electronmicroscopy were applied to mice vallate papillae. In normal mice, 1~2 serotonin immunoreactive cells were present in each taste bud section. After preloading 5-HTP, 3~6 cells showed strong immunoreactivity for serotonin. These cells were elongated, and their cytoplasm extended from the taste pore to the base of the taste bud. CGRP nerve fibers formed thick subgemmal nerve plexus under the basal lamina, and branched varicose perigemmal and intragemmal nerve fibers. Under the electron-microscope, three types of taste cells; dark cell, light cell and basal cells, were identified by their shape, location and electrical densities. Immuno-electronmicroscopy revealed that serotoninergic cells were dark cells. CGRP nerve fibers were located in and around taste buds, but the synaptic contacts with taste cells was not found. These findings indicate that mice taste cells are consisted of dark cell, light cell and basal cells, and dark cells contain serotonin. And, CGRP nerve fibers in taste buds may function as general sensory fibers.
5-Hydroxytryptophan ; Animals ; Basement Membrane ; Calcitonin Gene-Related Peptide* ; Calcitonin* ; Chromosome Pairing ; Cytoplasm ; Immunohistochemistry ; Mice* ; Nerve Fibers* ; Serotonin ; Taste Buds*

To evaluate the effect and working mechanism of a newly developed anti-cancer drug, AG60 (acriflavine-guanosine compound, Taerim Pharm. Co. Seoul, Korea), histotologic, autoradiographic and electron microscopic studies were carried out. For the histologic study, each Ehrlich carcinoma cells (10(7) cells)-inoculated mouse was subcutaneously injected with saline (0.2 ml), 10 mg/kg of AG60, or 30 mg/kg of AG60, every other day, respectively. Animals were sacrified on the 14th day from the first injection, and tumor masses were fixed in 10% formalin solution. Tissue sections of the tumor were stained with hematoxylin and eosin. For the electron microscopic study, Ehrlich carcinoma (10(7) cells)-inoculated mice were subcutaneously injected every other day with saline (0.2 ml) or 30 mg/kg of AG60, respectively. The day after 7th injection (14th day), animals were sacrified, small piece of tumor masses were fixed in 2.5% glutaraldehyde-1.5% paraformaldehyde solution followed by fixation in 2% osmium tetroxide solution. Ultrathin sections were counter stained with uranyl acetate-lead citrate solutions, and observed with JEM 100CX electron microscope. For the autoradiographic study, each Ehrlich carcinoma (10(7) cells)-inoculated mouse was injected every day with 0.2 ml of saline, 5 mg/kg of AG60, or 30 mg/kg of AG60, respectively. The day following the last injection, each animal was given a single dose of 0.7 micricurie/g of methyl-3H-thymidine (Amersham Lab., England) through the tail vein. Seventy minutes after the thymidine injection, animals were sacrified, tumor masses were collected and fixed in 10% neutral formalin. Tissue blocks were washed, dehydrated, embedded and cut in 6 micrometer-thick sections. Deparaffinzied sections were dipped in the autradiographic emulsion E1 (Amersham Lab., England) and dried and stocked in the dark room. Filmed sections were exposured five weeks in the dark room, and were developed in the developer. Labeled indices (mean number of labeled cells per 100 cancer cells) and labeled grain indices (mean number of labeled silver grains per one cancer cell, and total granule numbers per every 100 cancer cell) were observed and calculated. The results were as follows : 1. On histological study, massive apoptosis were occured following the injection of AG60. Only small number of live cancer cells were observed. 2. On electron microscopic study, massive apoptotic figures including fragmentation of nuclei and cytoplasms, multiple nucleoli, condensation of nucleus and cytoplasm, deep invaginations and microcleft formations of nuclei, margination of heterochromatin along the inner nuclear membrane and microcleft , etc. were noticed. Giant cells represent the "tumor cell-tumor cell emperipolesis", and many of them seem to be in process of "cytolytic emperipolesis". 3. On autoradiographic study, labeled grains of 3H-thymidine were suppressed to only 11%~5% of control cancer cells following AG60 administrations. Discussed on the above experiments, it is suggested that severe suppression of DNA, RNA and protein syntheses by AG60 induce massive apoptosis of cancer cells. AG60 is expected as one of most effective anticancer drugs for the cytostatic therapy, the disease stabilization, the improved quality of life, the prolongation of life, and possibly the chemoprevention.
Acriflavine ; Animals ; Apoptosis ; Autoradiography ; Edible Grain ; Chemoprevention ; Citric Acid ; Cytoplasm ; DNA ; Eosine Yellowish-(YS) ; Formaldehyde ; Giant Cells ; Guanosine ; Hematoxylin ; Heterochromatin ; Life Support Care ; Mice ; Microscopy, Electron ; Nuclear Envelope ; Osmium Tetroxide ; Quality of Life ; RNA ; Robenidine ; Seoul ; Silver ; Thymidine ; Veins

To observe the cellular expression of extracellular matrix components during mouse skin regeneration, the wounded skin samples were processed by immunoelectronmicroscopic methods, using primary antibodies for fibronectin, collagen type IV and laminin. The tissues were observed under transmission electron-microscope. The results were summarized as follows. 1. The granulation tissues and x-cells were observed in the wound margin at 18 hr post injury. The number of fibroblasts was increased in the granulation tissues at 1 day post injury. 2. The expression of fibronectin was observed in x-cells at 18 hr post injury, and in fibroblasts at 1 day post injury. In x-cells, after 1 day post injury, the expression of fibronectin was decreased. 3. At 1 day post injury, the expression of collagen type IV was increased in fibroblasts whereas not in x-cells. 4. The expression of laminin was increased by 18 hr post injury, but decreased after 1 day post injury. On the basis of above findings, in mouse, the regenerations of wounded skin were faster than other animals. The first step, infiltration response, was processed till 18 hr or 1 day post injury. The second step, fibroblast proliferation phase, began at 1 day post injury. In the regenerations of wounded skin, x-cells migrated to the wound region and activated, earlier than fibroblast. Thereafter, x-cells which appeared to be transformed into fibroblasts, played an important role in the synthesis of fibronectin and collagen type IV, and the formation of granulation tissue, with migrated fibroblasts to the wound region.
Animals ; Antibodies ; Collagen Type IV ; Extracellular Matrix* ; Fibroblasts ; Fibronectins ; Granulation Tissue ; Laminin ; Mice* ; Regeneration* ; Skin* ; Wounds and Injuries